In order to understand the development and functioning of the thymus, both in terms of T cell differentiation and stromal cell environmental support, we have undertaken a molecular approach to identify genes that are uniquely expressed in this organ. We have created an anchored RT- PCR based cDNA library from 14 day fetal thymuses after culturing them in deoxyguanosine and treating them with an anti-CD45 antibody to deplete the cell population of hematopoietic cells. The cDNA library was subtracted with poly A+ RNA prepared first from a fibroblast cell line and then from whole spleen. Three prime sequencing of 250 random cDNAs revealed 137 which were not in the databases of known sequences. The novel cDNAs were then screened by Northern blotting for expression in various tissues and in a set of SV40 transformed thymic epithelial cell lines. Four genes were selected for further analysis because they were limited in their expression to the thymus or to one of the stromal cell lines. All four were completely sequenced by obtaining full length cDNA clones from a SCID thymus library.During the past year the lab has made progress in understanding the structure and function of two of these genes. 1) The 1C12 gene, encoding a 12 transmembrane spanning co- transporter glycoprotein was disrupted by insertional mutagenesis using a DNA segment encoding both a beta galactosidase and a neomycin resistance gene. The insert was placed in the middle of the first exon. Heterozygous mice had very poor expression of the beta galactosidase gene and proved not be useful for identifying the cell type(s) expressing the 1C12 gene. Furthermore, homozygous gene-targeted mice showed normal thymic development as well as normal repopulation of the thymus in adult mice following sublethal irradiation. Thus, despite the exclusive expression of this protein in the subcapsular region of the thymus, no functional abnormalities were found by disrupting the gene. 2) The 1C10 gene has weak homology to transcription factors containing a POU domain and possesses several nuclear localization signals. To demonstrate that in fact the gene product goes to the nucleus, we engineered a 1C10 construct in which the gene for Green Fluorescent Protein was attached to the 3? end. GFP normally distributes in the cytoplasm, but when attached to 1C10 it accumulated in the nucleus. In a separate set of experiments we examined the expression of the 1C10 gene in thymus and lymph node in the absence of various lymphocyte cell populations, using a series of gene-targeted mutations. We found that expression in the lymph node required B cells, gamma delta T cells, and to a lesser extent alpha beta T cells, while expression in the thymus was independent of the presence of these cells and was even seen in RAG-/- mice which lack all but the earliest lymphoid precursors. Reconstitution of RAG-/- mice with normal bone marrow did not result in 1C10 expression. These observations suggest that 1C10 requires an inductive signal early in development in order to be expressed in the lymph node. - Thymus, genes, mice, knock-out, RT-PCR